I am trying to determine the reaction pathway for thymidilate synthase (TS). TS provides the sole de novo pathway for the biosynthesis of thymidine 5'-monophosphate (dTMP). As an essential enzyme for DNA synthesis, TS is found in almost all living species and many viruses. Thus, TS is an important drug target for the development of anti-cancer, anti-parasitic, anti-fungal, and anti-viral agents. TS uses methylene tetrahydrofolate as the carbon source for methylation of 2'-deoxyuridine 5'-monophosphate (dUMP) in the reaction. A species that offers great advantage in access to mutagenesis, and for which a wildtype crystal structure has been determined at UCSF, is L. casei (LCTS). Many kinetic studies on LCTS and mutants of the protein have been done in Dr. Santi's lab at UCSF. E. coli TS (ECTS) mutants are now also being studied. I have completed studies on the crystal structures of ternary and binary complexes of the poorly active LCTS mutant E60Q. I am continuing studies on three other mutants that greatly affect enzyme activity (D221A, D221C, and R218K) and have extended study to E. coli TS mutant ternary complexes involving D169C. The graphics computers and software at Computer Graphics Laboratory have been extremely helpful in these studies. With them, I have been able to compare directly the active sites of the wildtype and mutant LC/ECTS ternary complexes in order to deduce crucial changes in important water molecule positions and amino acid sidechain orientations that may affect enzyme activity.